Bacterial pathogens of both plants and humans use a type III secretion system to inject effector proteins into the cells of their hosts. As many as 30 or more different 'type III effectors' can be delivered by an individual pathogen and once inside the host cell they manipulate various processes to promote bacterial virulence. The great diversity of type III effectors, and the fact that their amino acid sequences offer few clues to their function, has been an impediment to understanding how they manipulate the host. One host process, the attachment of ubiquitin to a protein in order to regulate its function or to cause its degradation, plays an important role in host immunity and offers a vulnerable target for type III effectors. This application concerns the characterization of a type III effector, AvrPtoB, from the plant pathogen Pseudomonas syringae. The crystal structure of a C-terminal domain of AvrPtoB revealed that it is a structural mimic of the U-box class of E3 ubiquitin ligases - a type of host protein that facilitates the attachment of ubiquitin to other proteins. The AvrPtoB E3 ligase is able to use host E2 conjugating enzymes to cause ubiquitination and degradation of a host protein kinase required for immunity. Despite these advances, much remains to be learned about how this bacterial E3 ligase binds and ubiquitinates its host substrates in order to subvert their normal functions. Recently virulence proteins from human pathogens such as Legionella pneumophila and Escherichia coli have also been found to express proteins of the U-box class of E3 ligases. Structural mimicry of this type of host protein therefore appears to be a common strategy for undermining host immunity. The long term objective of this project is to increase our understanding of the underlying mechanisms that bacterial E3 ligases use to manipulate host ubiquitination. This knowledge will be useful for the development of novel strategies for interfering with these virulence proteins in order to lessen the impact of pathogen infection in both plants and humans. The goals of this application are to: 1) Identify the host E2 conjugating enzyme used by AvrPtoB and characterize the role of a ubiquitin-interaction motif that lies close to the U-box; 2) Exploit the recently solved crystal structure of AvrPtoB in complex with one of its host targets to understand how AvrPtoB recognizes and binds its substrates; 3) Perform a screen to identify additional host proteins that AvrPtoB ubiquitinates and which are candidates for playing a role in host immunity; 4) Investigate the way in which certain host-mediated modifications of AvrPtoB (e.g. phosphorylation) influence its E3 ligase activity; and 5) Develop a method to visually monitor the delivery of AvrPtoB into the host cell. The method will be used to test the hypothesis that type III effectors that act early in the infection process are delivered before later-acting effectors. PUBLIC HEALTH RELEVANCE: Manipulation of host immunity by a bacterial E3 ubiquitin ligase Gregory B. Martin, Boyce Thompson Institute for Plant Research at Cornell University Project Narrative This research will elucidate how a bacterial virulence protein, which is injected into host cells, interferes with the host immune response. The knowledge gained will contribute to strategies to lessen the impact of bacterial diseases of importance to both agriculture and human health.